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Abstract:

Mumps virus (MuV) is an enveloped, non-segmented, negative-sense, RNA virus, genus
Paramyxoviridae. MuV causes an acute, systemic infection in humans characterised by parotitis,
orchitis and aseptic meningitis. Despite widespread vaccination, outbreaks continue to occur.
The biology of MuV infection and pathogenesis in humans remains poorly understood, in part
due to a lack of physiologically-relevant virus and host cell model systems. Of particular interest
is how MuV interacts with epithelial cells during infection. Therefore, a reverse genetics system
was generated based on the consensus genomic sequence of unpassaged, clinical material from
a genotype G MuV infection from the New York 2009 outbreak. The resulting viruses were
referred to as recombinant (r) MuVG09. Establishment of this reverse genetics system allowed
the generation of rMuVG09 expressing the enhanced green fluorescent protein (EGFP) in the
third position, rMuVG09EGFP(3) to allow real -time tracking of virus replication in vitro.
Surprisingly, recovered viruses had mutations, suggesting genetic instability of MuVG09 during
growth in vitro. However, using this system, it was demonstrated that MuV could infect
transformed and primary polarised epithelial cells from the apical and basolateral surfaces.
Infection of differentiated, normal, human, bronchial, epithelial (dNHBE) cells showed that MuV
could infect ciliated and non-ciliated, non-goblet cells. MuV was released from the apical and
basolateral surfaces. In dNHBE cells this was associated with cell -to-cell spread and cell -to-cell
fusion. MuV cell -to-cell fusion was controlled by haemagglutinin-neuraminidase (HN) and fusion
(F) glycoproteins. Mutation of a single amino acid (I278T) in the F glycoprotein ablated cell -to-cell
fusion while preserving virus-to-cell fusion, which was associated with increased virion stability.
Analysis of growth kinetics in vitro showed that the non-fusogenic virus was growth restricted in
interferon (IFN) competent cell lines. This was correlated with an increase in interferon stimulated
gene (ISG) expression during infection, suggesting a role for fusogenicity in evading the human
IFN response.